Division Procedure:X1
United Kingdom
- 1..
ELISA plates are coated with 50 μl of rabbit antiviral sera overnight in a humidity chamber at room temperature.
- 2..
Fifty microlitres of a duplicate, twofold series of each test serum starting at 1/4 are prepared in U-bottomed multiwell plates (carrier plates). Fifty microlitres of a constant dose of antigen are added to each well and the mixtures are left overnight at 4 °C. The addition of the antigen reduces the starting serum dilution to 1/8.
- 3..
The ELISA plates are washed five times with PBST.
- 4..
Fifty microlitres of serum/antigen mixtures are then transferred from the carrier plates to the rabbit-serum-coated ELISA plates and incubated at 37 °C for one hour on a rotary shaker.
- 5..
After washing, 50 μl of guinea-pig antiserum to the antigen used in point 4 is added to each well. The plates are incubated at 37 °C for one hour a rotary shaker.
- 6..
The plates are washed and 50 μl of rabbit anti-guinea-pig immunoglobulin conjugated to horseradish peroxidase is added to each well. The plates are incubated at 37 °C for one hour on a rotary shaker.
- 7..
The plates are washed and 50 μl of orthophenylene diamine containing 0,05 % H 2 O 2 (30 %) w/v is added to each well.
- 8..
The reaction is stopped after 15 minutes with 1,25M H 2 SO 4 .
The plates are read spectrophotometrically at 492 nm on an ELISA reader linked to a microcomputer.
For each antigen used 40 wells contain no serum but contain antigen diluted in PBST. A duplicated twofold dilution series of homologous bovine reference antiserum. A duplicate twofold dilution series of negative bovine serum.
Antibody titres are expressed as the final dilution of tests serum giving 50 % of the mean OD value recorded in the virus control wells where test serum is absent. Titres in excess of 1/40 are considered positive.
Hamblin C, Barnett ITR and Hedger RS (1986) ‘ A new enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against foot-and-mouth disease virus. I. Development and method of ELISA. ’ Journal of Immunological Methods, 93, 115 to 121.11.